This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Due to their sessile habit, plants have evolved a complex set of secondary metabolites to protect them from diseases, herbivory and many other environmental stresses. We are employing a combined protein crystallographic/biochemical-genetics approach to understand the details of structures and functions of the key biosynthetic enzymes that underlie the diversity of secondary metabolism. Our current focus involves a group of S-adenosylmethionine (SAM) dependent O-methyltransferases, enzymes that facilitate methylation of polyphenolic (iso)flavoniods (flavonoids and isoflavonoids). Specific methylated forms of (iso)flavonoids have been shown to mediate many processes including resistance to UV irradiation, fungal pathogens, insect attack, and plant-rhizobium signaling for N2 fixation. We recently identified a small family of (iso)flavonoid O-methyltransferases (IOMTs) from the legume model plant Medicago truncatula. However the structural underpinnings governing their biochemical properties are not completely understood, and the catalytic roles these enzymes play in planta remain to be defined. We propose a combined X-ray crystallographic and mutagenesis approach to explore the structural basis for their regiospecific and stereoselective methylations. We also propose to use reverse genetics and metabolic profiling to assess the in vivo functions of the specific IOMT family members for (iso)flavonoid biosynthesis. These structure and function analyses will lead to a more complete understanding of the molecular basis that underlies the complexity of (iso)flavonoid biosynthetic pathways.